Traditional plunger displacement pumping systems have been successful in delivering stable, accurate flows in the normal-scale and micro-scale high performance liquid chromatography (HPLC) regimes. While normal-scale HPLC is performed with mobile phase flow rates of about 0.1–5.0 mL/min and micro-scale HPLC is performed with mobile phase flow rates of about 1–100 μL/min, nano-scale HPLC requires mobile phase flow rates in the 50–500 nL/min range. Plunger displacement pumping systems can not deliver nano-scale HPLC flow rates with reliability and accuracy.
One method for providing nano-scale flow rates in an HPLC system is to use a flow-divider which directs a majority of flow from the pump to a waste stream and a small portion of the pump output to the HPLC working stream (i.e., to the liquid chromatography column). A split restrictor in the waste stream and/or the working stream controls the split ratio of the system. Normal-scale or micro-scale HPLC pumps can be used in split flow mode to produce nano-scale HPLC flow rates in the working stream.
In order to operate an HPLC system in split-flow mode the user must calculate the split ratio of the system. To calculate the split ratio, the user must know the permeabilities of both the split restrictor and the chromatographic system (i.e. the packed column). These permeabilities are used to calculate the flow rate that must be supplied by the normal-scale or micro-scale HPLC pump to produce the desired flow through the chromatographic system. Although it is possible to calculate split restrictor dimensions that should provide a desired split ratio, changes in permeability of either the split restrictor or chromatographic column over time cause unpredictable split ratio variations. Such variations result in unacceptable flow variations through the chromatographic column.
One possible solution to the problem of changing split ratios is to monitor the flow to the chromatographic column with an appropriate flow sensor. Fluid flow rates can be determined by measuring the pressure of a liquid flowing through a restrictor. Assuming a constant viscosity, the back pressure of liquid flowing through a restrictor will scale linearly with the flow rate of the liquid. The flow rate is measured by placing a pressure transducer before and after a restrictor inline with the flow. Signals from the pressure transducers are electronically subtracted and amplified to achieve a high degree of common-mode noise rejection.
The permeability of the restrictor is chosen so that it provides sufficient back pressure to produce a measurable pressure difference signal (ΔP) in the flow ranges of interest but does not produce a significant back pressure for the pump. For example, a 10 cm long, 25 μm inside diameter capillary will provide a back pressure of approximately 100 pounds per square inch (psi) for water flowing at 5 μL/min. This permeability is sufficient for providing a flow measurement while not inducing much fluidic load on the pump.
However, pressure measuring flow sensors must be calibrated to compensate for the different viscosity of each fluid being measured. This creates a great disadvantage in liquid chromatography applications wherein fluid composition varies dramatically over the course of a chromatography run.
Another method that can be used to sense fluid flow is thermal flow sensing. Several companies including Sensirion AG, of Zurich, Switzerland, and Bronkhorst Nijverheidsstraat of Ruurlo, The Netherlands, have been developing thermal flow sensors capable of monitoring flows in nL/min ranges.
Operation of these thermal flow sensors is described with reference to FIG. 1. Heat introduced into a liquid filled tube/channel will disperse in both the upstream and downstream directions (i.e. due to thermal conduction or diffusion respectively). The tube of the flow sensing device is made from materials of low thermal conductivity (i.e. glass, plastic). A temperature profile similar to curve “A” in FIG. 1 will develop when a discrete section of the fluid in the tube is continuously heated, under a zero flow condition. The shape of this temperature profile will depend upon the amount of heat added to the fluid and the upstream and downstream temperatures of the liquid. Assuming identical upstream and downstream fluid temperatures, under a zero-flow condition, liquid temperatures measured at P1 and P2 will be equal as thermal diffusion will be equal in both directions.
If the liquid in the tube is permitted to flow, the fluid temperatures at P1 and P2 will depend upon the rate of liquid flux and the resulting heat convection. As liquid begins to flow past the heated zone, a temperature profile similar to curve B in FIG. 1 will develop. In addition to the symmetric diffusion of the heat, asymmetric convection of the heated fluid will occur in the direction of the fluid flow. Therefore, under flowing conditions, fluid temperatures measured at P1 and P2 will be different.
Temperature measurements made at P1 and P2 are sampled, subtracted and amplified electronically in situ to provide a high degree of common-mode noise rejection. This allows discrimination of extremely small upstream and downstream temperature differences. By appropriate placement of temperature measurement probes (i.e., P1 and P2) and/or by changing the amount of heat added to the flowing liquid, temperature measurement can be made at inflection points along the temperature profile. Measurement at the inflection points maximizes the upstream/downstream ΔT response to flow rate change.
The dynamic range of thermal flow sensing is limited by the sensitivity and precision of the temperature measurement instruments. Heat transfer via diffusion in the fluid and heat transfer to the tube walls occurs rapidly under low fluid flux conditions (i.e. <500 nL/min in tubing/channels of dimensions <100 um). Therefore, precise measurement of temperature close to the point of heat addition is required. The upper dynamic range is limited by the dynamic range of the temperature sensors and by the amount of heat that can be added to the flowing liquid.
However, like pressure measuring flow sensors which must be calibrated to compensate for the different viscosity of each fluid being measured. Thermal based flow sensors also need such calibration. This creates a great disadvantage in liquid chromatography applications wherein fluid composition varies dramatically over the course of a chromatography run.